This invention includes retrovirus envelope mutants into which heterologous peptide or glycopeptide sequences can be fused for expression and stable presentation on retroviral, viral and liposome vectors. The invention further relates to methods of making and using these retrovirus envelopes for gene and drug therapy.
1. Gene Therapy Vectors
Numerous gene therapy vectors have been created. These vectors are constantly being engineered to overcome problems caused by tropism, infectivity and virus stability. Engineered gene therapy virus vectors include adenoviruses (see as examples, D. Armentano et al., 1998 U.S. Pat. No. 5,707,618; T. J. Wickham et al., 1998 U.S. Pat. No. 5,731,190; and M. Cotten et al., 1997 U.S. Pat. No. 5,693,509), herpes simplex viruses (R. L. Martuza et al., 1998 U.S. Pat. No. 5,728,379) and retrovirus vectors. Non-viral vectors include episomal and liposomal vectors such as those described in M. J. Cooper, 1997 U.S. Pat. No. 5,624,820; and L. Li et al., 1997 U.S. Pat. No. 5,641,508.
Retroviruses have been preferred vectors for gene therapy based on their ability to integrate retroviral DNA into the genome of the host cell. Retroviral gene therapy patents include vectors with multiple cloning sites (M. Eglitis et al., 1997 U.S. Pat. No. 5,672,510), retroviruses with mixed LTRs (H. M. Temin et al., 1996 U.S. Pat. No. 5,554,524), retroviruses that deliver genetic elements that stimulate an immune response (H. E. Gruber et al., 1998 U.S. Pat. Nos. 5,716,826 and 5,716,426), and vectors with specific envelope proteins (E. F. Vanin et al., 1998 U.S. Pat. No. 5,710,037).
2. Retroviral Envelope Mutations and Gene Therapy
The retrovirus envelope protein is the viral element that allows a gene therapy vector to recognize and bind to the target, typically a host cell. The native or wild-type retrovirus envelope protein has a natural tropism for certain target cells, which typically must be overcome if the virus is to be used as a gene therapy vector. However, the envelope protein engineered to overcome wild type tropisms must nevertheless maintain the characteristics of: (1) surface (SU) protein stability such that it is not shed from the virus particle too quickly; and (2) infectivity wherein the virion can infect the host cell and introduce the genetic material it is carrying.
The envelope protein in all retroviruses is produced as a glycoprotein precursor that matures into two cleavage products: the surface protein (SU) and transmembrane protein (TM). TM and SU are held together by disulfide bonds and perhaps other non-covalent interactions (J. N. Coffin et al., 1997 Retroviruses Cold Spring Harbor Press). Envelope shedding, in Moloney murine leukemia virus (MoMLV) for example, occurs as result of the weak linkage created by the disulfide bonds between the TM (p15E) and the SU (gp70). Although mutations have been made to prevent cleavage of the envelope precursor protein in MoMLV gp80 into SU and TM, typically there has been an associated loss of incorporation into virions observed with these mutants making them undesirable vectors (E. O. Freed et al., 1987 J. Virol. 61: 2852-6). Finally, in HIV, the endoproteolytic cleavage of the envelope precursor protein has been demonstrated to be required for the activation of HIV (J. M. McCune et al., 1988 Cell 53: 55-67).
Envelope proteins also are known to possess highly conserved functional domains. In MoMLV, amino acid residues 1-33 constitute the leader sequence; amino acids 34-263 constitute the receptor binding domain; amino acids 264-312 comprise the hinge region; and residues 313-469 constitute the body portion of the surface protein (J. M. Mason et al., 1997 U.S. Pat. No. 6,643,770). Mutagenesis analysis of the envelope protein has led to the discovery of other amino acid residues that appear responsible for receptor binding (A. J. MacKrell et al., 1996 J. Virol. 70: 1768-74) and fusion events (Y. Bae et al., 1997 J. Virol. 71: 2092-9). Although at least one group has proposed that the N-terminal 72 residues of the amphotropic 4070A isolate are not required, for amphotropic receptor usage (C. Peredo et al., 1996 J. Virol. 70: 3142-52), other researchers have demonstrated that the N-terminus of the envelope protein is required especially when preparing fusion envelope proteins (see for examples, F-L. Cosset et al., 1995 J. Virol. 69: 6314-22; S. Valsesia-Wittmann et al., 1996 J. Virol. 70: 2059-64; and J. M. Heard et al., 1991 J. Virol. 65: 4026-32). Additional mutagenic analysis of retrovirus (e.g., in PVC-211 murine leukemia virus and MoMLV) envelope proteins has been described and is discussed in the following: M. Masuda et al., 1996 J. Virol. 70: 8534-9; and A. J. MacKrell et al., 1996; and H. Skov et al., 1993 J. Gen. Virol. 74:707-14).
3. Fusion Glycoproteins
One method of overcoming the retrovirus"" natural tropism is by expressing an envelope fusion glycoprotein. A fusion glycoprotein contains the retroviral envelope (env) protein, e.g., the SU protein, linked to a selected peptide or glycopeptide. For examples, see R. W. Paul et al., 1998 U.S. Pat. No. 5,736,387 and S. J. Russell et al., 1998 U.S. Pat. No. 5,723,287. Many of the engineered envelope proteins created to target the retrovirus particle to other cells comprise insertions of heterologous peptides into the amino terminus of SU (F-L Cosset et al., 1995 J. Virol. 69: 6314-22). Fusion glycoproteins have been developed to compensate for the folding problems created due to changes in the glycosylated pattern. (S. Kayman et al., 1997 U.S. Pat. No. 5,643,756).
The inventors disclose novel mutant envelope proteins which when linked or fused to a heterologous peptide or glycopeptide have enhanced stability and maintain retrovirus virion titer and infectivity levels comparable to that observed for wild type retrovirus envelope proteins. Due to the ability to restore the target penetration capability that is lost or greatly diminished upon fusion of a heterologous sequence into the envelope protein, vectors containing these mutant envelope proteins have an increased ability to penetrate targets, typically cells, and a correspondingly increased ability to deliver nucleic acids or drugs. Further, due to the increased association between SU and TM the mutant envelope proteins are more stable and vectors containing these mutant envelope proteins are less likely to shed off of the vector or infective particle. Decreased envelope shedding increases the life span of a virion so it can survive such mechanical stressors as freezing and thawing or vascular shearing forces. Correspondingly, vectors containing these mutant envelope proteins would be extremely useful as nucleic acid and drug delivery vehicles. Methods of identifying retrovirus mutant envelope proteins possessing these desirable characteristics based on three-dimensional structural motifs are also disclosed.
This invention discloses isolated nucleic acid molecules encoding retrovirus envelope proteins or polypeptide fragments thereof having decreased shedding of binding sequences through the suppression of envelope protein cleavage comprising an amino acid substitution in at least one amino acid of at least one of seven motifs or corresponding amino acid residues in other retrovirus envelope proteins. The first five (5) motifs as described for MoMuLV which exhibit this function comprise the following amino acids: (1) 104Lys, 107Glu, 90Thr, 102Arg and 108Thr; (2) 124Arg, 138Tyr, 128Ser, 132Gly, 134Pro, 121Gly, and 133Gly; (3) 223Arg, 225Arg, 224Leu, 16Glu, 24Thr, and 201Thr; (4) 137Phe, 135Asp, 136Ser, 208Arg, and 217Gly; and (5) 142Trp, 152Trp, 210Tyr, 141Tyr and 151Tyr. For motif 6, which comprises 227Gln, 228Asn, and 243Asp, the isolated nucleic acid molecule encodes a retrovirus envelope protein or polypeptide fragment thereof having increased penetration capability through restoration of the function of residue 8His and decreased shedding of binding sequences through stabilization of SU:TM interaction comprising an amino acid substitution in at least one of the listed amino acid residues. For the seventh motif, the envelope protein encoded by the nucleic acid molecule has increased penetration capability arising from a substitution in at least one of the residues comprising the motif containing: 198Ser, 11Tyr, 226Tyr, 35Trp, 38Trp, 196Val, 197Thr, 160Tyr, 158Trp, 123His, 203His, 233Val, 235Ile, 240Val, 241Leu, and 8His. The amino acid substitutions contemplated for the residues of these seven motifs are listed in Table I. A short hand form for delimiting said motifs 1-7 is denoted by amino acids 104Lys, 124Arg, 223Arg, 137Phe, 142Trp, 227Gln and 198Ser, respectively. Substitution of amino acids comprising other retroviral envelope proteins which are in alignment with the above amino acids comprising the 7 motifs are also contemplated. Such an alignment of amino acids comprised in the retroviral envelope proteins for a variety of retroviruses are displayed in the alignments as set forth in FIGS. 15-17.
The inventors also disclose a retrovirus envelope protein that is encoded by the described nucleic acid molecules.
The retrovirus envelope proteins and fragments thereof and the nucleic acid molecules encoding said envelope proteins described above are derived from: Moloney MLV; Friend MLV; MLV 10A1; MLV 4070A; AKV MLV; CasBrE; RadLV; MCF1233; Xeno CWM; Xeno NZB; feline leukemia virus types A and B (FeLV-A and FeLV-B); avian leukosis retrovirus; GALV (gibbon ape leukemia viruses) SEATO strain; and human immunodeficiency virus type 1 (HIV-1). The residue or residues in one or more of the motifs to be altered are determined by alignment of the MoMLV envelope protein sequence with one of these other retrovirus sequences. Alignment of the residues of each of the motifs for these retroviruses is depicted in FIGS. 15-17. Similar alignments can be generated for human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) envelope proteins using the Megalign software of DNASTAR.
The invention also discloses recombinant retrovirus particles that infect eukaryotic cells comprising one of the mutant envelope proteins or polypeptide fragments described above and a nucleic acid encoding said envelope protein or polypeptide fragment thereof. Eukaryotic cells contemplated for infection include vertebrate cells, mammalian cells and human cells. The envelope proteins of these recombinant retrovirus particles may further comprise a heterologous polypeptide displayed on the external surface of the particle wherein the heterologous peptide is fused to the retroviral envelope protein or polypeptide fragment thereof.
The invention contemplates a producer cell line transduced with one of the above described retrovirus particles, as well as retroviruses particles produced from said producer cell line. These retrovirus particles can also be utilized to transduce eukaryotic cells, such as vertebrate cells, mammalian cells and human cells.
The inventors also disclose packaging cell lines comprising one of the described nucleic acid molecules.
The inventors further disclose methods of delivering a nucleic acid molecule to treat a disease or condition comprising the step of administering to a subject a retrovirus particle or liposomal particle which comprises at least a retrovirus mutant envelope protein or polypeptide fragment thereof as described above.
The mutant envelope proteins and polypeptide fragments described above can be utilized in liposome compositions, pseudotype viruses, and pseudotype retroviruses, including combinations with lipid destabilizers. A method of administering these compositions to directly and selectively deliver an agent to a target cell is also contemplated.